5. Processing

Apply zero-filling to 128 real data points in F) and to 128 real data points in F to obtain a matrix of 1024«128x128 real data points. This would result in a huge file of processed real data. Since only the amide proton region is of interest, use strip transformation in F3 (350 points). Application of forward linear prediction (20 coefficients) for both F, and F) results in somewhat better resolved 3D spectra. Use Gaussian multiplication [gb = 0.1, lb = –3 Hz] in F; and a 7/2-shifted squared sinusoidal window in the other dimensions. For the data file described, extensive experimentation with different window functions may be worthwhile. Phase correction and base-line correction may be necessary for all dimensions. Further details are very dependent on the particular software you use to process such a 3D data file. Reference the ¹⁵N dimension (F2) using the =-scale procedure described in the introduction of this chapter.

6. Result

The figures show two planes of the 3D spectrum obtained on a DRX-600 spectrometer with a multinuclear inverse z-gradient triple resonance probe-head (fixed third channel on BC), As in the HCCH-TOCSY (Exp. 15.12) we choose isoleucine I-61 as an example, since TOCSY is most important for amino acids with a long aliphatic side chain. In a an expansion of the H,H plane with d, = 118.6 as parameter is shown. The NH proton of I-61 has its signal at 64 = 7.25, Hp at dy = 1.39, H, at dy = 1.09 and the two methyl groups at 6, = 0.46 and 0.41. In contrast to the result of the HCCHTOCSY (see Exp. 15.12) the resolution here in the indirect proton dimension is insufficient to separate the signals of Hg and H, and those of Hy and Hs. The signals of the second proton strip in a at dy = 7.48 arise from arginine R-54 with dj = 119.1, which is seen in the same plane due to the limited resolution in F2. The proton signals of H, at 5, = 4.71 and of the B,y, and 6 protons are seen at dj = 2.2 to 1.8.

As a second example we show in b an H,H plane chosen at 6 = 132.2 as parameter.

Here the very simple TOCSY spectrum of alanine A-46 with 5, = 9.0 is shown, demonstrating a typical application of this method, an easy identification of alanine